Electrophoresis is a phenomenon in which charged particles move in a conductive buffer medium or fluid across which a potential difference is applied. The migration is toward an electrode carrying charge opposite to that of the particles.
Electrophoresis is one of the most important methods available for the investigation of biological materials, and probably the most efficient procedure for the separation and detection of proteins and other matter.
Electrophoresis separation relies on the differential speeds of the migration of differently charged particles in an electrical field. The migration speed is primary a function of the charge on the particle and the field strength applied and the charge on a particle is determined by the pH of the buffer medium. The most important application of this technique in biomedical research and clinical chemistry laboratories, is in the electrophoretic separation of proteins, nucleic acids, their component peptides and oligonucleotides, as well as complex macromolecules such as lipoproteins.
Several different systems are known for practicing electrophoretic separation. For example, one system, known as zonal procedures, has advantages but it also has certain limitations. Some of the most common limitations are: The amount of sample required in order to reveal the components by the common staining procedures is usually large, the preparation of the apparatus and complete system involved in the electrophoretic separation is commonly tedious and time consuming, the time required to obtain complete separation of the components is often hours, the time required to reveal the components and to obtain some quantitation of the separated substances is also commonly hours, the yield of recovery of the components as biological actives in most cases is very low, the reproducibility of the electrophoretic separation is not 100 percent accurate, and the automation to perform the entire system operation is almost lacking.
Capillary electrophoresis has been shown to be a technique for obtaining high separation efficiency. For some proteins and small peptides, separation efficiencies of approximately one million to about a few million have been demonstrated. In general, this technique utilizes a fused silica (quartz) capillary with an inside diameter ranging from about 25 microns to about 200 microns, and a length ranging from about 10 centimeters to about 100 centimeters. Since the entire volume of the column is only 0.5 to about 30 microliters (yielding probably the smallest total surface area of column chromatography), the injection volume is usually in the low nanoliters range. As a consequence, the sensitivity of this technique is quite high and it is possible to obtain quantitation in the order of picomoles (and probably femtomoles or attomoles) using fluorescence, electrochemical, laser induced fluorescence, and mass spectrometry detectors, and to obtain quantitation in the order of nanomoles using ultraviolet detectors.
In capillary electrophoresis, the efficient heat transfer from small diameter capillaries permits application of unusually high voltages ranging from about 5,000 volts to about 30,000 volts while maintaining a low current, in the range of about 10 microamperes to about 90 microamperes. The application of high voltages promotes more effective separations and increases the speed of analysis to record times of about 5 to 40 minutes.
In addition to high separation efficiency (theoretical plates), fairly high resolution, high sensitivity quantitation, and small migration (retention) times, capillary electrophoresis presents a few more advantages over conventional electrophoresis, and in general, other chromatographic procedures. Some of these advantages are: a) application to a wide variety of samples ranging from small ions to proteins or other macromolecules of molecular weights of approximately 290,000 daltons or higher (such as DNA fragments, viruses, and subcellular particles) by using essentially the same column and probably the same conditions of electrophoretic separation; b) capillaries should provide an ideal system to explore nonaqueous media, particularly with substances which are highly hydrophobic; c) capillaries are reusable many times making the electrophoretic separation system very practical and economical; d) on-line electronic detection permits good quantitation and further enhances possibilities for fully automatic operation making the capillary electrophoresis system of higher resolution, greater speed, and better accuracy than conventional methods.
In the prior art, it is generally known that a material, containing mixtures of substances to be analyzed, can be passed along a capillary tube and through a detector under the influence of an applied voltage. The applied voltage charges the substances and the charges on the substances determine their spacing and their speed of passage along the capillary tube.
The prior art, U.S. Pat. Nos. 3,620,958, 3,948,753 and 4,459,198, show electrophoresis apparatus including a capillary tube connected between two containers for containing the substance to be analyzed and having electrical potential applied between the two containers and across the capillary tube. While the various forms of apparatus shown in these patents are apparently useful, they require large concentrations of samples to be analyzed and none is capable of being automated or provides teaching related to automation.
The present invention provides high voltage capillary electrophoresis apparatus including, among other things, means for feeding small concentrations of sample material into a capillary tube, automatically applying the proper voltage to cause the components of the sample to be charged and to flow along the capillary tube through a detector wherein the components are detected and a printed record is made. The apparatus can then automatically repeat the process for the analysis of multiple samples.
The basic apparatus of the invention is susceptible of many modifications in its various parts including the capillary tube portion. In addition, the method of detection of samples may be varied and the collection of samples can be modified. The invention can also be adapted to measure electroosmotic flow in a capillary.